Every year there are new chemicals introduced into the occupational and environmental settings, which together with those already present may increase the risk of different adverse health effects. Epidemiological data clearly shows an increase in the prevalence of immunological disorders, which in part can be related to xenobiotic exposure as for example is in the case of allergic diseases, including asthma, among populations of industrialized countries. The immune system consists of very sensitive and specific network of cellular and humoral interactions that, when deregulated, causes the general malfunction of physiological processes of the host. Immunotoxicity is understood as the ability of a given compound to alter function of immune system of human or animal in a deleterious way.
Immunotoxicity testing is difficult and it is rather generally accepted that it cannot be accomplished with a single test. Strategies for immunotoxicity testing that have been developed and underwent the process of validation are based on a battery of tests. Frequently a “two tier” approach is proposed for immunotoxicity testing. In such a system the first tier consists of several screening tests detecting general abnormalities in the immune system such as morphological changes, while the second tier represents a more in depth evaluation of immune function following contact with given compound. Most of the tests for immunotoxicity that have been developed and validated employed experimental animals. These tests use a combination of in vivo, ex vivo and in vitro assays of immune functions and frequently involve isolated immune cells, usually lymphocytes. The difficulties related to predictive testing for immunotoxicity are in part related to multiple molecular and cellular targets of immunotoxin actions that have to be taken into account.
WO99/37142 concerns the production of transgenic animals for the study of Insulin-dependent Diabetes Mellitus. The document describes a method for producing a transgenic non-human mammal carrying a transgene encoding an immuno-inducible autofluorescent protein, said method comprising chromosomally incorporating a first DNA sequence encoding a cytokin promoter operatively connected to a second DNA sequence encoding said autofluorescent protein, such as Green Fluorescent Protein (GFP) or its enhanced variants (EGFP) into the genome of a non-human mammalian animal. A somatic cell from the transgenic mammal has been claimed inter alia.
WO02/22786 describes a cell line comprising a human cell line capable of producing a selected cytokine associated with an inflammatory response in humans, and transfected with a vector containing DNA encoding a cytokine regulatory factor (CRF) under the control of a promoter, and a vector containing DNA encoding a detectable-marker protein, under the control of a promoter responsive to cytokine induction. Disclosed cell line is useful for screening test compounds for anti-inflammatory activity, by its culturing under conditions in which CRF is overproduced in the transfected cells, the selected cytokine is induced, and the detectable-marker protein is produced at detectable levels, adding a test compound to the cultured cells, and observing any diminution in the level of the detectable-marker protein. Furthermore, an amount of dsRNA effective in stimulating cytokine production in the cytokine overproducing cells is added to the culture, and a priming agent such as phorbol myristate acetate (PMA), calcium ionophores, sodium butyrate, endotoxin, and cytokines is also added. WO00/75660 provides methods of screening a test agent for the ability to reduce osteoclastic bone reabsorption. In a preferred embodiment, the methods involve screening the agent for the ability to inhibit tumor necrosis factor (TNF-alpha) expression through activity at an inhibitory TNF-alpha-responsive element (TNF-Re) in the tumor necrosis factor promoter or through activity at a complex formed by an estrogen receptor at TNF-Re. The document discloses screening a test agent for the ability to modulate osteoclastic bone reabsorption comprising: (a) contacting an estrogen receptor (ER) and a gene under the control of a tumor necrosis factor (TNF) modified promoter with a test agent; and (b) detecting a difference in the level of gene expression compared with a control cell. The method is useful for screening agents that reduce osteoclastic bone reabsorption, and for the identification of compounds that modulate TNF-alpha expression resulting in reduced osteoclastic bone reabsorption.
W00050872 provides systems, methods, screens, reagents and kits for an optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions.
WO0023615 discloses a method for extracting quantitative information relating to an influence on a cellular response in mechanically intact or permeabilized living cells, which method comprises recording variation in spatially distributed light emitted from a luminophore as a change in light intensity, measured by an instrument designed for the measurement of changes in fluorescence intensity. The luminophore, which is present in the cells, is capable of being redistributed in a manner which is related with the degree of the influence, and/or of being modulated by a component which is capable of being redistributed in a manner which is related to the degree of the influence.
There are attempts of in vitro immunotoxicity testing using isolated immune cells. In such tests immunosuppressive activity is expected when proliferative response or physiological functions like NK activity of immune cells are downregulated, and immunostimulation is expected when upregulation of these responses is detected. The possible immunomodulatory action is especially difficult to assess in vitro because of our limited understanding of molecular and cellular mechanisms mediating this effect. A specific problem for using in vitro tests lays in the fact that they generally do not take into account possible roles of metabolism in the xenobiotic's action.
Cytokines are generally defined as proteins secreted by cells that affect the behavior of other cells. They are produced by many cell types and through specific receptors affect activity of cells of different origin. It is believed that cytokines are critical regulators that orchestrate immune response by interconnecting dispersed elements of the immune system into one functional entity. They are grouped into families: the hematopoietins, the interferons, the chemokines, and the TNF family. T cells, among which one can distinguish function-related diversity, produce the greatest proportion of cytokines.
There is more and more evidence showing that at least certain types of immunotoxicity, such as those leading to hypersensitivity and autoimmunity are associated with modulation of the expression of particular cytokine genes in immune cells or non-immune cells. This effect seems to be important for immunotoxicity associated with heavy metals and amino acid derivatives linked to Eosinophilia-Myalgia Syndrome (USA in 1986). Derivatives of amino acids, which were putative ethiological agents in the epidemic mentioned above, were also shown to induce expression of a potent immunomodulatory cytokine, IL-5, in immune cells cultured in vitro. IL-4, the cytokine critical for development of allergic response, was secreted by lymphocytes and mast cells following contact with heavy metals, which represents a good correlation of xenobiotic action in vitro (upregulation of IL-4 in immune cells) and in vivo (upregulation of IgE in experimental animals). Certain cytokines expressed by non-immune cells are also important signals modulating immune response. For example, some chemical allergens were reported to stimulate keratinocytes to express particular cytokines such as IP-10, MIP-2, IL-1β, and IL-10. Some types of immunosuppression can also involve the modulation of cytokine expression like that observed with azathioprine or cyclosporin A, which inhibit IL-2 expression in lymphocytes.
In light of the presented current state of technology, it is desirable to develop new methods of (immuno)toxicity testing in vitro, which would not only indicate perturbations of the immune system but also allow the elucidation of the potential mechanism of immunomodulation. This approach would utilize the knowledge about pleiotropic activities of cytokines that regulate different processes of immune system.
Thus, the main objective of this invention is a development of a new system of characterising the biological activities of xenobiotics in vitro. A particular goal of the invention is to facilitate easy and reliable tests for their toxicity, particularly immunotoxicity.